The use of combinatorial methods in both chemistry and biochemistry over the last few years has generated an enormous number of (bio)molecules of pharmaceutical (drugs) and biotechnological (biocatalysts) interest. The ability to effectively identify active molecules in random libraries relies on the availability of a screening method able to select the positive variants or derivatives that can be further characterised and commercialised. An ideal, general screening method would be able to select the positive targets from a random library of molecules/substrates tested against a specific binding protein/enzyme, as well as to select positive, active variants of a random library of binding proteins/enzymes against a specific target molecule/substrate.
It is desirable to identify agents that can modulate NAD(P)H and/or an NDO. This is because NAD(P)H is a ubiquitous cofactor required by many oxidoreductases of biotechnological interest. NAD(P)H-dependent oxidoreductases are widely spread in nature, carrying out a great number of reactions that can be of biotechnological interest. The availability of a screening method that would allow for workers to identify potential substrates for these heterogeneous class of enzymes, or for the activity of a library of random mutants with different specificities towards selected targets, would be of great importance for engineering purposes. In particular, cytochromes P450 are widespread enzymes in nature involved both in biosynthetic and biodegradation pathways. The wide range of their functions and specificity presents attractive possibilities for their use in different applications, such as large scale chemical synthesis and drug metabolite production, toxicity screening tests of drugs or chemicals as well as detoxification of waste and in situ bioremediation of contaminated areas (1).
The present invention seeks to provide an effective assay method for identifying agents that could modulate NAD(P)H and/or an NDO (or even vice versa).